Katopodis et al. Journal of Ovarian Research (2021) 14:84 https://doi.org/10.1186/s13048-021-00834-9

RESEARCH Open Access p38β - MAPK11 and its role in female cancers Periklis Katopodis1,2* , Rachel Kerslake1 , Athanasios Zikopoulos3, Nefeli Beri4 and Vladimir Anikin2,5

Abstract Background: The p38MAPK family of Activated Protein are a group of signalling molecules involved in cell growth, survival, proliferation and differentiation. The widely studied p38α isoform is ubiquitously expressed and is implicated in a number of cancer pathologies, as are p38γ and p38δ. However, the mechanistic role of the isoform, p38β, remains fairly elusive. Recent studies suggest a possible role of p38β in both breast and endometrial cancer with research suggesting involvement in bone metastasis and cancer cell survival. Female tissue specific cancers such as breast, endometrial, uterine and ovary account for over 3,000,000 cancer related incidents annually; advancements in therapeutics and treatment however require a deeper understanding of the molecular aetiology associated with these diseases. This study provides an overview of the MAPK signalling molecule p38β (MAPK11) in female cancers using an in-silico approach. Methods: A detailed expression and methylation analysis was performed using datasets from cBioportal, CanSar and MEXPRESS. Breast, Uterine Endometrial, Cervical, Ovarian and Uterine Carcinosarcoma TCGA cancer datasets were used and analysed. Results: Data using cBioportal and CanSAR suggest that expression of p38β is lower in cancers: BRCA, UCEC, UCS, CESC and OV compared to normal tissue. Methylation data from SMART and MEXPRESS indicate significant probe level variation of CpG island methylation status of the gene MAPK11. Analysis of the ’ two CpG islands shows that the gene was hypermethylated in the CpG1 with increased methylation seen in BRCA, CESC and UCEC cancer data sets with a slight increase of expression recorded in cancer samples. CpG2 exhibited hypomethylation with no significant difference between samples and high levels of expression. Further analysis from MEXPRESS revealed no significance between probe methylation and altered levels of expression. In addition, no difference in the expression of BRCA oestrogen/progesterone/HER2 status was seen. Conclusion: This data provides an overview of the expression of p38β in female tissue specific cancers, showing a decrease in expression of the gene in BRCA, UCEC, CESC, UCS and OV, increasing the understanding of p38β MAPK expression and offering insight for future in-vitro investigation and therapeutic application. Keywords: MAPK11, MAPK, Female cancers, Breast cancer, Ovarian cancer, Cervical cancer, Uterine carcinosarcoma, Meexress, Smartapp, Cbioportal, canSar, Methylation, p38β, Pan-cancer

* Correspondence: [email protected] 1Biosciences, College of Health and Life Sciences, Brunel University London, Uxbridge, UK 2Division of Thoracic Surgery, The Royal Brompton & Harefield NHS Foundation Trust, Harefield Hospital, London UB9 6JH, UK Full list of author information is available at the end of the article

© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Katopodis et al. Journal of Ovarian Research (2021) 14:84 Page 2 of 12

Introduction as signalling molecules such as the Mitogen Activated The establishment of cancer as one of the leading causes Protein (MAPK) family kinase proteins [11]. of morbidity and mortality globally is widely accepted, yet the complexity of molecular processes that contrib- MAPKs ute to the development and progression of the numer- Mitogen activated protein kinases (MAPKs) play vital ous subsets of cancer continues to plague researchers. roles in signalling transduction pathways and ability to Of the 18.1 million incidences of cancer worldwide in control intracellular processes such as cell survival, dif- 2018 over 20% of cases are thought to be female repro- ferentiation, proliferation and apoptosis, via the sequen- ductive tissue specific [1]. The most frequently diag- tial phosphorylation of substrate protein Ser/Thr kinase nosed type of female specific cancers is breast cancer protein cascades. The three-tiered activation structure with 2,088,849 cases recorded in 2018, around only 1% consists of the activation of a MAPK Kinase Kinase of which are male [2]. Cervical cancer is the most fre- (MAP 3 K) followed by the phosphorylation of a MAPK quently diagnosed of the gynaecological cancers, with Kinase leading to the dual phosphorylation of MAPK 569,847 cases diagnosed annually followed by endomet- proteins [12]. The conventional subfamilies of MAPK in- rial and uterine with 382,069 cases; ovarian is the least clude: the extracellular signal regulated kinase (ERK) common yet deadliest with 295,414 cases diagnosed glo- proteins, ERK1/2 and ERK5 along with the stress acti- bally each year and over 184,779 deaths [1]. vated Jun amino-terminal kinases (JNK) as well as the Knowledge regarding associated risk factors such as stress activated p38MAPK proteins (Fig. 1)[12, 13]. diet, weight, heritability, and menopausal status along with global health initiatives, routine cervical screening, p38β and breast imaging, have led to earlier detection of both The p38MAPK family is comprised of four homologous cervical and breast cancer [3, 4]. However, recurrence proteins: p38α, p38β, p38δ and p38γ and is involved in and mortality rates are still troublesome. Gynaecological the integration of biochemical signals in response to en- cancers such as uterine and endometrial are also often vironmental stresses as well as reactive oxygen species detected at early to mid-stages with treatment for female (ROS) and inflammatory cytokines [14, 15]. The reproductive tissue cancers often involving surgical pro- p38MAPK family has been implicated in a number of cedures such as complete or partial mastectomy (breast) cancer pathologies. p38α is the most widely studied of or hysterectomy [5]. Ovarian cancer, however, is more the isoforms and is ubiquitously expressed along with frequently diagnosed at an advanced stage owing to the p38β, of which it shares around 75% homology, whereas ambiguous nature of symptoms related to many gynae- p38δ and p38γ are differentially expressed throughout cological disorders [6]. Despite comprising the lowest the tissues [16]. number of gynaecological cases, ovarian cancer is the Over the last few decades, evidence implicating regula- deadliest of gynaecological malignancies with five-year tion of p38 with processes involved in cancer such as survival less than 26% for those diagnosed with advanced epithelial-mesenchymal transition, migration, invasion, serous ovarian cancer [7]. Notwithstanding the contin- and survival has grown. Until recently, investigation of ued advancements in detection and treatments of cancer p38 had primarily focused on the biomarker p38α, how- many of the molecular mechanisms implicated in the de- ever mounting evidence also suggests roles for p38β and velopment, progression and resistance related recurrence the other isoforms [17–20]. of female reproductive tissue specific cancers require p38β is encoded by 12 exons of the gene MAPK11 that further investigation [8]. lie on 22 at the position 22:55,263,713-50, Of the multitude of risk factors associated with the de- 270,380 (UCSC genome browser/GRCh38/hg38). It is velopment of cancer, hormone dysregulation is substan- comprised of 364 amino acids and contains a kinase do- tial in breast, with the onset of menopause lowering the main comprised of a T-G-Y dual phosphorylation motif risk of breast cancer while prolonged exposure to en- that enables activity [21]. p38β is expressed in the vast dogenous hormones such as oestrogen as and oestrogen majority the organs and cell types with high expression receptor status presenting an increased risk [9]. In in endothelial cells yet low in those of hematopoietic ori- addition, age, as well as genome instability and mutation gin such as macrophages and monocytes [22]. p38β is are often implicated in the development and progression activated by MKK6 and expressed at a lower basal level of cancer. Heritable mutation of the DNA repair genes while, p38α is activated by MKK3/6 often leading to the BRCA1 and BRCA2 are not only significantly associated past characterisation of p38β as redundant [23]. p38α with breast cancer but are also biomarkers of ovarian expression is crucial for foetal development and its ab- and endometrial cancer [10]. Other notable genes in- sence is lethal, while expression of p38β is not [24–26]. clude the Tumour Suppressor protein 53 (TP53) as well Both p38α and p38β, along with the downstream as angiogenic factors such as VEGF and TGF-B as well MAPKs MK2/3/5, act as key regulators in gene Katopodis et al. Journal of Ovarian Research (2021) 14:84 Page 3 of 12

Fig. 1 a MAPK11 protein association network acquired by StringDB; b MAPK signalling pathways showing the initiation of JNK and p38 following external stimulation from UV and Reactive Oxygen Species (yellow) as well as CKs and growth factors (red). The p38 isoforms (green) are phosphorylated via MKK3/6 and MKK4 (grey) in response to ROS and environmental stress consequently increasing activation of downstream targets involved in processes such as cell migration, survival and differentiation, proliferation and apoptosis. UV: Ultraviolet radiation; ROS: Reactive Oxygen Species; GFs: Growth Factors; CKs: Cytokines; MEKK3,4: MAP3K3, MAP3K4; TAO: Serine/threonine-protein kinase Tao; ASK: Apoptosis signal- regulating kinase, MAP3K5; MLK1–3: Mitogen-Activated Protein Kinase Kinase Kinase 9–11; MKK4: Mitogen-Activated Protein Kinase Kinase 4; STAT1/3: Signal Transducer And Activator Of Transcription 1/3; ATF1,2: Activating 1,2; cMYC: MYC Proto-Oncogene; ELK1: ETS Transcription Factor ELK1 expression as well as cell cycle progression and are acti- overview of the role of p38β in all cancers with a de- vated in response to stress and many pathological pro- tailed focus of p38β in female tissue specific cancers. cesses including inflammation and cancer [27]. Unlike p38α, p38β modulates basal activity through self- p38β in female cancers activation via autophosphorylation [28]. While a lot of Breast cancer (BRCA) work has been done on the p38α expression and activity, p38MAPKs translate extracellular signals to intracellular the roles of p38MAPKs (β/γ/δ) remain to be elucidated. response. Their role in cell proliferation, differentiation Recent literature has presented emerging roles for and response to inflammation is vital for normal cell p38β in tumour progression including the ability to function. They are regulators of major pathways includ- regulate TGF-1β and VEGF growth factor mediated sur- ing the ERK1/2-JNK (extracellular signal regulated- vival of endothelial cells in the absence of the proapop- kinase, c-Jun N-Terminal Kinase) pathways which are. totic p38α [29]. both associated with poor clinical outcome in breast − − In addition, p38β is shown to play a significant role in cancer patients [38]. Despite the viability of a p38β / the regulation of the oncogene lipocalin 2 (LCN2) a tar- knock out mice and the consideration of p38α as the get of plakophilin 3 (PKP3). Where PKP3 expression is predominant isoform in response to cellular stresses decreased p38β is capable of regulating LCN2 leading to there is increasing evidence of p38β involvement in an increase in tumour invasion and metastasis [30]. In stress response and cancer cell biology [39, 40]. Upregu- all cancers, an increasing number of key proteins or long lation of p38β in breast cancer is a prime example, p38β non-coding RNAs are also starting to be associated with regulation may not have been linked to tumour growth p38β expression and protein-protein interaction [31– however increased expression is associated with upregu- 35]. The Long non-coding RNA, LINCO1220 in particu- lation of monocyte chemotactic protein-1 (MCP-1) lead- lar, has received interest due to its roles in p38β sup- ing to osteoclast differentiation and promotion of bone pression and in endometrial cancer and as such its metastasis in breast cancer patients [40]. Apart from the therapeutic potential [33]. following gynaecological cancers, although rare (~ 1%), Despite the numerous studies investigating the expres- breast cancer may affect men as well. Male breast cancer sion and role of p38β in cancers such as lung, Head and is often associated with inherited mutations of BRCA1, Neck Squamous Cell carcinoma as well as prostate the BRCA2 and PIK3CA genes, obesity, estrogen treatments role in female tissue specific cancers remains elusive [36, (hormonal therapies for prostate cancer) and Klinefelter 37]. As such, this bioinformatic review will provide an syndrome [41–43]. Katopodis et al. Journal of Ovarian Research (2021) 14:84 Page 4 of 12

Ovarian cancer (OV) female malignancies this absence presents an area for It has been found that Galectin-1 (Gal-1) is an indicator further investigation. of poor prognosis in ovarian cancer patients where high expression of Gal-1 is observed in ovarian cancers of Cervical squamous cell carcinoma (CESC) higher histological grade as well as advanced lymph node In Cervical Squamous Cell Carcinoma, activation of p38 status [44, 45]. Activation of the MAPK JNK/p38 path- when driven by mediators such as osteopontin (OPN) way is influenced by Gal-1 and facilitates epithelial- leads to invasive progression. Phosphorylated p38 by mesenchymal transition (EMT) making it a promising CD44-mediated MKK3/6, high expression of OPN and target for prevention of epithelial ovarian cancer metas- furin, induction of NF-κB and p65 are correlated with tasis [45]. In addition to Gal-1, overexpression of genes cervical cancer progression and are considered thera- such as LAMC2 are also associated with increased cell peutic targets [55]. Not only is p38 inhibition a target of proliferation, repression of cell apoptosis and increased novel anti-tumour agents, there are also reports of sup- expression of p38. Inhibition of p38 nuclear accumula- pression through use of traditional herbal medicines tion through the negative regulation of LAMC2 by miR- such as Matrine, which consequently also decreases the 125a-5p is also shown to repress tumorigenesis in epi- expression of matrix metalloproteinases, MMP-2 and thelial ovarian cancer [46]. MMP-9 [56]. In contrast, activation of MMPs via p38/ NFκB pathway with inflammatory cytokines like inter- Uterine Corpus endometrial carcinoma (UCEC) leukin 17A (IL-17A), is associated with the invasion of cervical cancer cells, making IL-17A another potential Uterine Corpus Endometrial Carcinoma has not been in- prognosis marker of CESC [57]. Despite the tumorigenic vestigated in as much detail, but a few markers have nature of p38 there is evidence of dual role status as been identified that play significant roles in the progres- both a tumour suppressor and protooncogene; Xantho- sion of this type of cancer. Mutation of the PTEN gene, noids such as Alpha-mangostin for example elevate re- for example, affects cellular signalling through inhibition active oxygen species (ROS) as well as p38 and of MAPK pathway and is associated with histological consequently damage mitochondrial integrity while insti- subtype as well as the early stage characteristics of endo- gating apoptosis within cervical cancer cells [58]. metrial cancer. Regulation of cell proliferation - differen- Using large-scale data sets such as TCGA, genetic ana- tiation by MAPK via RAF-MEK-ERK may provide a lysis and potential biomarker identification is becoming targetable mechanism for future research [47]. Studies increasingly accessible to researchers. In-silico analysis have shown that oestradiol is a potential activator of the using online datasets and tools can further the under- MAPK pathway through estrogen (ER) and Insulin re- standing of alterations and mechanisms which influence ceptor (InsR) interaction. Insulin synergistically with ER expression such as the mutational status and methyla- activates phosphorylation of MAPK and PI3K pathways tion profile of genes of interest without the need of ex- [48, 49]. Phosphorylated Akt (p-Akt), mTOR (p-mTOR) pensive and time consuming in-vivo/in-vitro analysis. and MAPK (p-MAPK) proteins as well as lncRNAs like This research seeks to use data visualization tools com- HEIH, are often implicated in endometrial carcinogen- piling data from TCGA for the analysis of the p38MAPK esis and tolerance of common antineoplastic agents such isoform p38β in cancers of female reproductive tissue as paclitaxel [50, 51]. As such, MAPK and PI3K/Akt are origin, presenting evidence for the further exploration of promising targets of new anti-tumor agents such as em- this signalling molecule in cancer cell biology for mo- odin (rhubarb) [47]. lecular understanding and potential therapeutic translation. Uterine Carcinosarcoma (UCS) Uterine carcinosarcoma is usually classified as uterine Results endometrial carcinoma but is often more aggressive than The PanCancer analysis of MAPK11 provides an over- UCEC. Little is known about the molecular effects that view of expression throughout the early and advanced influence aggressive biphasic growth of the sarcomatous stages of the disease as well as the genetic alterations of elements that account 2–5% of the uterine corpus malig- MAPK11 that contribute towards changes in expression. nancies [52, 53]. It has been shown that UCSs undergo Focusing on female tissue-specific cancers (Fig. 2a), we EMT and highly express signal transducers such as outline a trend where MAPK11 expression in normal tis- TGF-β; SMAD2/3, playing an important role in the sue is higher than that of early-stage BRCA, CESC, regulation of cell growth and development [54]. There is UCEC, and UCS cancer data sets. Unlike the other fe- very little evidence regarding investigation of MAPK and male cancers, MAPK11 expression in OV is primarily other signalling pathways in UCS within the literature, shown at the advanced stage of the disease which is in light of the involvement of MAPK signalling in other often the stage of diagnosis due to the ambiguity of Katopodis et al. Journal of Ovarian Research (2021) 14:84 Page 5 of 12

Fig. 2 (See legend on next page.) Katopodis et al. Journal of Ovarian Research (2021) 14:84 Page 6 of 12

(See figure on previous page.) Fig. 2 a PanCancer expression analyses of MAPK11 from canSAR database. A higher expression is seen in normal (blue) samples from female data sets: BRCA, OV, UCEC, and UCS compared with early (yellow) and advanced stage (pink) samples, with OV exhibiting the lowest level of expression in the advanced stage compared to all cancer data sets; b PanCancer data analysis using cBioPortal provides an overview of the landscape of MAPK11 genetic alterations across the range of TCGA data sets. OV, UCEC exhibit the highest level of alteration within the cancer types with deep deletion presenting as the most prevalent throughout the data sets. Of the female-specific cancers: BRCA, OV, CESC, UCEC, each exhibit varying levels of amplification specific alterations as well as high levels of deep deletion; c Deep deletions in BRCA 7/1084, CESC: 4/297, OV: 28/585, UCEC: 22/529 and UCS: 1/57. OV and the UCEC patients with deep deletions of MAPK11 are the biggest populations compared to all other cancers; d MAPK11 expression in female tissues: BRCA, CESC, UCEC, and UCS, indicating higher expression in normal tissue compared to downregulation in cancer data sets across all four cancer stages symptom presentation (Fig. 2d). The average expression with the N-Shore probe cg03717414, with lower methy- of MAPK11 in OV (0.9 RSEM (log2(value + 1)), is rela- lation in all tested cancers compared to normal and a tively low compared to the other female cancers and is positive correlation between expression and methylation, mirrored in the low expression alteration profile seen in again with exception to UCS (R = -0.0053). Following the Fig. 2b where shallow and deep deletion are the most genomic region of MAPK11, we found 10 locations on 2 frequent type of alteration. islands with the first island having 4 methylation targets (CpG1: cg23755154, cg15036874, cg16054907, and methylation cg13577505) to present a strong positive correlation of It is generally considered that DNA methylation within expression-methylation for all 4 cancers (exception is the CpG island region of a gene’s promoter region is probe cg15036874 in BRCA with an R = -0.095). Follow- correlated with gene expression while DNA methylation ing the S-shore region, the next 6 locations located within the gene body is also associated with chromo- within the second island exhibit hypomethylation with a somal integrity [59]. To analyse the methylation status strong correlation between expression and methylation of MAPK11, 14 probes were chosen through the SMAR status (CpG2: cg19184963, cg08211722, cg15554007, T database and studied within the female cancers: cg00735239, cg00164898, cg00395632). Detailed results BRCA, CESC, and UCEC (Table 1). are located in Additional files 1, 2, and 3. Differences be- Here, we find a positive correlation of MAPK11 ex- tween the observed levels of expression and region- pression with methylation of the promoter region. Two specific methylation are seen in Fig. 3 may influence the of these CpG islands were found within the genomic re- binding of transcriptional factors at the promoter region gion of MAPK11. The probe cg26790091 at the S-Shelf of the gene as well as the stability of the gene itself [60]. region showed lower methylation values compared to Gene expression and methylation analysis show that in normal datasets and a positive correlation of expression BRCA, methylation at cg23755154 is significantly in- and methylation was seen for all datasets with the excep- creased while the hypomethylated cg19184963 and tion of UCS (R = -0.04). The same pattern is observed cg00395632 are decreased within the tumour dataset

Table 1 List of the 14 probes analysed Probe Chromosome Start End CGIposition CpG cg26790091 chr22 50,264,031 50,264,032 S_Shelf cg03717414 chr22 50,265,420 50,265,421 N_Shore cg23755154 chr22 50,266,978 50,266,979 Island CpG1 cg15036874 chr22 50,267,519 50,267,520 Island cg16054907 chr22 50,267,636 50,267,637 Island cg13577505 chr22 50,267,819 50,267,820 Island cg06018119 chr22 50,268,221 50,268,222 S_Shore cg19184963 chr22 50,270,210 50,270,211 Island CpG2 cg08211722 chr22 50,270,495 50,270,496 Island cg15554007 chr22 50,270,507 50,270,508 Island cg00735239 chr22 50,270,518 50,270,519 Island cg00164898 chr22 50,270,698 50,270,699 Island cg00395632 chr22 50,270,792 50,270,793 Island cg11070772 chr22 50,272,060 50,272,061 S_Shore Katopodis et al. Journal of Ovarian Research (2021) 14:84 Page 7 of 12

Fig. 3 a Genomic information of the gene MAPK11. The segment plot showing the detailed information of genomic locations of CpGs of MAPK11, highlighting CpG island, shelves, and shores. The name and the type of each transcript are given. The coverage of the CpG islands is displayed as the red region. 14 probes are included in this genomic location with 10 of them to lie on the two island regions (CpG1 and CpG2); b Red bars on indicate hypermethylated regions. MAPK11 gene can be found in the right end of the chromosome at the cytogenetic band 22q13.33 (green box)

(Additional file 1). In CESC, the dataset exhibits signifi- island region yet hypomethylated in the second for both cant hypermethylation at cg15036874 and cg16054907 cancer data and normal, there is little significant differ- along with hypomethylation of the aggregated probes ence between the levels of expression and methylation with tumour showing higher levels of methylation com- for the aggregated probes of BRCA with CESC and pared to normal tissue (Additional file 2). In UCEC, sig- UCEC only exhibiting slight variation. Further analysis nificant probe sites include the hypermethylation of of the collection of probes aligning at the CpG island re- cg23755154 and cg16054907 (Additional file 3). Infor- gions of the gene MAPK11 in wildtype and cancer data mation about the UCS and OV aggregation could not be sets can be seen in Additional Fig. 5. obtained due to limitations of the SMART analysis tools Complementary methylation analysis using MEX- (see Additional file 4). PRESS, indicates that there is no significant difference in We chose to group the methylated probes by region the expression of the MAPK11 between the stages of separating those that are located at CpG island 1 and each cancer data set studied (Fig. 5, Additional file 5). those at CpG island 2 to analyse the separate promoter Moreover, no significant changes were observed for the binding regions. A Spearman correlation analysis be- BRCA oestrogen and BRCA progesterone receptor sta- tween the expression of MAPK11 and DNA methylation tus, HER2/neu receptor, or menopause status of the can- (M value) of MAPK11 in three of the female cancers in- cers (data not shown here). dicates that expression is significantly positively associ- ated with the methylation of region 1, a phenomenon Methods that does not align with the normal paradigm of gene MEXPRESS web tool was used to integrate TCGA data expression (cg23755154, cg15036874, cg16054907, for the visualisation of gene specific methylation and ex- cg13577505). Negative correlation however was seen pression of MAPK11 in relation to genomic location and with the following 6 probes that are localised to the sec- clinical data such as age, weight and receptor status [61]. ond CpG island: cg19184963, cg08211722, cg15554007, DNA methylation is a reversible process consisting of cg00735239, cg00164898, cg00395632 (Fig. 4, see Add- the covalent binding of a methyl group to cytosine, one itional files 1, 2, and 3). of four DNA bases. In humans, DNA methylation is al- Average aggregation of probes indicates that the gene most exclusively restricted to the cytosines of CpG dinu- is hypermethylated within the region of the first CpG cleotides and plays a critical role in the regulation of Katopodis et al. Journal of Ovarian Research (2021) 14:84 Page 8 of 12

Fig. 4 MAPK11 Methylation and correlation of expression with methylation in a BRCA; b CESC; c UCEC. In the top, the CpG1 island with 4 probes is showing a clear positive correlation of the expression of MAPK11 with the methylation status, while at the bottom, the CpG2 island with 6 probes, is showing a clear negative correlation of the expression with the methylation. Overall, a high methylation status has been observed in the first 4 positions targeted by the probes and lower methylation was observed in the following island, indicating that the gene is split into 2 genomic regions of different methylation status (see more at Additional files 1, 2 and 3)

Fig. 5 MEXPRESS view of the TCGA data for MAPK11 in breast invasive carcinoma. The samples are ordered by MAPK11 expression, revealing that the expression in the different stages is not significantly altered. Probes have been coloured differently depending on the genomic region they target. The detailed analysis for all cancers analysed can be found in Additional file 5 Katopodis et al. Journal of Ovarian Research (2021) 14:84 Page 9 of 12

gene expression. Abnormal DNA methylation patterns within the MAPK11 gene were the most frequent type are found ubiquitously in all types of human cancer. The of alteration within the gene and no significant differ- precise genomic location of DNA methylation is one of ence of expression seen between stages of the cancers the most important regulatory factors of gene expres- studied (TCGA data regarding OV is not as extensive as sion, and is where MEXPRESS distinguishes itself from the other data sets). These trends in the expression are other available tools [62]. converse to that of p38α which is often over-expressed Shiny Methylation Analysis Resource Tool (SMAR within cancer cells and considered to render the roles of T) provided access to a comprehensive overview of p38β redundant. However, recent publications suggest MAPK11 DNA methylation and associated omics data, that p38β is capable of self-activation through autophos- through integration of TCGA data [63, 64]. This applica- phorylation and the regulation of specific pathways out- tion was used to correlate epigenetic modifications with lined in the introduction [28, 72]. gene expression in cancer data sets using probes specific Regulation of genes is often influenced through methy- to genomic regions of MAPK11. The following 14 lation. Using the SMART analysis tool, the methylation probes were used in analysis of MAPK11 (Τable 1). status of MAPK11 at specific regions was assessed. The cBioPortal (www.cbioportal.org/) which con- Grouped analysis of the methylation probes specific to tained both sequencing and pathological data on 30 dif- the two CpG islands located on the MAPK11 gene was ferent cancers was used to analyze the genetic alteration used to reveal the methylation status and expression of of MAPK11 across different cancer types [65, 66]. The the gene promoter regions. Hypermethylation of the Breast Invasive Carcinoma (TCGA, PanCancer Atlas, gene in the CpG1 was seen for BRCA, CESC, and UCEC n = 1075), Cervical Squamous Cell Carcinoma (TCGA, with an increase in the methylation of the gene in PanCancer Atlas, n = 297), Ovarian Serous Cystadeno- tumour samples as well as a positive correlation with ex- carcinoma (TCGA, PanCancer Atlas, n = 585), Uterine pression. This trend does not follow the general para- Corpus Endometrial Carcinoma (TCGA, PanCancer digm often seen with hypermethylation and gene Atlas, n = 529), Uterine Carcinosarcoma (TCGA, Pan- suppression suggesting that methylation is not the only Cancer Atlas, n = 57) datasets were selected for further regulator of gene expression and that there are add- analyses of MAPK11 expression and mutation status itional transcriptional and post-transcriptional factors [65, 66]. influencing the decreased levels of p38β expression canSAR. Pan-cancer analysis of the MAPK11 expres- within these cancers. The CpG2 island however displays sion and expression in the different cancer stages of the hypomethylation of the region along with negative cor- four female cancers was conducted through canSAR relation and relatively high levels of gene expression with (cansar.icr.ac.uk), a public, freely available, integrative little difference between cancer and normal. translational research and drug discovery knowledge The differential methylation of specific regions of a base [67]. gene is often associated with both expression and gene stability. Therefore, the different levels of methylation Discussion within the CpG islands of MAPK11 between normal and The MAPK signalling cascades are integral signalling cancer data sets may influence the decrease in expres- mechanisms that play an active role in cellular processes sion of MAPK11 seen in cancer tissues and warrants fur- including proliferation, apoptosis, differentiation, and ther investigation as a possible biomarker (Figs. 2, 3, 4) cell development. The p38MAPK isoforms, in particular, [60]. mediate response to external stresses, inflammatory cy- Additional analysis through MEXPRESS was also used tokines as well as reactive oxygen species (ROS) [68–70]. to assess the correlation between the receptor status and Until recently, clinical evaluation of a family of key expression of MAPK11. The oestrogen receptor alpha is regulators, the p38MAPK isoforms have widely focused thought to mediate the development of endometrial can- on the study and consideration of p38α as a biomarker cer and is a biomarker of breast cancer. However, no dif- of cancer [71]. However, recent evaluations of p38 impli- ferences in expression were seen relative to oestrogen, cate the additional isoforms with cancer cell biology. progesterone, or HER2/neu receptor status as well as The expression of p38β has received growing interest for menopausal status suggesting that p38β activation is not its roles in female cancers such as breast and endomet- mediated via these pathways that are often associated rial [33]. with female cancers (Additional file 5)[48]. Data generated within this study sought to explore the Despite the decreased level of expression, the high expression of p38β in the female tissue-specific cancers: level of deep deletion within the gene and differences in BRCA, CESC, OV, UCEC, and UCS, revealing down- methylation may influence post-transcriptional regula- regulated expression of the gene in each cancer data set tion and phosphorylation of the gene, influencing its compared to normal tissue (Fig. 2). Deep deletions ability to self-activate. For example, therapeutics such as Katopodis et al. Journal of Ovarian Research (2021) 14:84 Page 10 of 12

emodin which target phosphorylation are shown to methylation. Overall, a high methylation status has been observed in the maintain basal levels of genes involved in the p38MAPK first 4 positions targeted by the probes and lower methylation was pathway while inducting ROS activated apoptosis of can- observed in the following island, indicating that the gene is split into 2 genomic regions of different methylation status. cer cells [47, 73]. β Additional file 3:. MAPK11 Methylation and correlation of expression Activation of p38 via the transcription factor Poke- with methylation in UCEC. In the top, the CpG1 island with 4 probes is mon/Zbtb7 is associated with tumorigenesis and cell in- showing a clear positive correlation of the expression of MAPK11 with vasion in HepG2 cells [30]. Downstream signalling of the methylation status, while at the bottom, the CpG2 island with 6 β probes, is showing a clear negative correlation of the expression with the p38 also leads to an increase in expression of the onco- methylation. Overall, a high methylation status has been observed in the gene, Lipocalin2 (LCN2), and an increase in tumour cell first 4 positions targeted by the probes and lower methylation was migration [17, 30]. Additional downstream targets in- observed in the following island, indicating that the gene is split into 2 genomic regions of different methylation status. clude mediators of proliferation and survival such as Additional file 4:. MAPK11 Methylation and correlation of expression AP-1 and mTOR which consequently are also involved with methylation in BRCA, CESC, UCEC and UCS of all the 14 probes. in gynaecological malignancy [74, 75]. MAPK11 has Additional file 5:. Detailed presentation of methylation status of the shown oncogenic properties through increased regula- MAPK11 gene in BRCA, CESC, UCEC and UCS as retrieved from tion of the WNT inhibitor, Dickkopf WNT signalling MEEXPRESS analysis. The CpG island probes are presented in red and asterisks annotations represent statistical significance. pathway inhibitor 1 (DKK-1), a key regulator of bone Additional file 6:. Heatmap of all 14 methylated MAPK11 probes in metastasis [31]. Elevated levels of DKK-1 are also associ- BRCA, CESC, UCEC and UCS. The CpG island probes are presented in red. ated with advanced clinical stage and poor prognostic outcomes in gynaecological malignancies including ovar- Acknowledgements ian cancer [76]. The upregulation of p38β through We thank Miss Heerni Halai for helping with the acquisition of some of the lncRNA 1220 is also associated with endometrial cancer figures and proofreading. through the mediation of proliferation and inhibition of β Authors’ contributions apoptosis. p38 phosphorylates Myocyte Enhancer Fac- Periklis Katopodis had roles in conceptualization, methodology, data tors (MEF) such as MEF2A and MEF2C regulators of curation, funding acquisition, investigation, visualization, supervision, analyse differentiation, proliferation, apoptosis, migration as well of all data sets, formal analysis, acquiring the figures, writing-original draft, α reviewing, and editing. Rachel Kerslake had roles in investigation, writing- as metabolism especially in the absence of p38 [77]. It original draft, and writing-reviewing and editing. Athanasios Zikopoulos and has been shown that it increases osteoclast differenti- Nefeli Beri had roles in writing-reviewing. Vladimir Anikin had roles in review- ation and activity through upregulation of monocyte ing and funding acquisition. All authors read and approved the final manuscript. chemotactic protein-1 (MCP-1) [40, 78]. It has also been suggested that the p38MAPKS are capable of acting as Funding both a tumour suppressor as well as an oncogene [79– No funding was received for this publication. 81]. With the lower expression levels, its ability to regu- Declarations late these vital cell functions and act as a tumour sup- pressor may be compromised. Ethics approval and consent to participate Further investigation of the role in these female tissues Not applicable. may provide an opportunity for uncovering a duel role Consent for publication of MAPKp38 isoforms as both oncogenes and tumour Not applicable. suppressors in cancer cell biology. Competing interests The authors declare that they have no competing interests. Supplementary Information The online version contains supplementary material available at https://doi. Author details org/10.1186/s13048-021-00834-9. 1Biosciences, College of Health and Life Sciences, Brunel University London, Uxbridge, UK. 2Division of Thoracic Surgery, The Royal Brompton & Harefield 3 Additional file 1:. MAPK11 Methylation and correlation of expression NHS Foundation Trust, Harefield Hospital, London UB9 6JH, UK. Obstetrics with methylation in BRCA. In the top, the CpG1 island with 4 probes is and Gynaecology Department, Royal Cornwall Hospitals NHS Foundation 4 showing a clear positive correlation of the expression of MAPK11 with Trust, Royal Cornwall Hospital, Truro TR1 3LJ, UK. Department of Medicine, 5 the methylation status, while at the bottom, the CpG2 island with 6 Karolinska Institutet, 17177 Stockholm, Sweden. Department of Oncology probes, is showing a clear negative correlation of the expression with the and Reconstructive Surgery, Sechenov First Moscow State Medical University, methylation. Overall, a high methylation status has been observed in the Moscow, Russian Federation 119146. first 4 positions targeted by the probes and lower methylation was observed in the following island, indicating that the gene is split into 2 Received: 25 November 2020 Accepted: 7 June 2021 genomic regions of different methylation status. Additional file 2:. MAPK11 Methylation and correlation of expression with methylation in CESC. In the top, the CpG1 island with 4 probes is References showing a clear positive correlation of the expression of MAPK11 with 1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide the methylation status, while at the bottom, the CpG2 island with 6 – probes, is showing a clear negative correlation of the expression with the for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68(6):394 424. https://doi.org/10.3322/caac.21492. Katopodis et al. Journal of Ovarian Research (2021) 14:84 Page 11 of 12

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